RESUMEN
We hypothesized that the scarcity of N-nucleotides might contribute to the inability of the neonate to mount a robust allergic immune response. To test this, we used terminal deoxyribunucleotidyl Transferase deficient (TdT-/-) mice, which express "fetal-like" T cell receptor and immunoglobulin repertoires with largely germline-encoded CDR3 regions. Intraperitoneal sensitization was followed by aerosol provocation with either PBS or the allergen OVA in both TdT-/- mice and wild-type mice to develop allergic respiratory inflammation. The effects of this procedure were investigated by lung function test, immunological analysis of serum and brochoalveolar lavage. The local TH2 cytokine milieu was significantly attenuated in TdT-/- mice. Within this group, the induction of total IgE levels was also significantly reduced after sensitization. TdT-/- mice showed a tendency toward reduced eosinophilic inflow into the bronchial tubes, which was associated with the elimination of respiratory hyperreactivity. In conclusion, in a murine model of allergic airway inflammation, the expression of fetal-like antigen receptors was associated with potent indications of a reduced ability to mount an asthma phenotype. This underlines the importance of somatically-generated antigen-receptor repertoire diversity in type one allergic immune responses and suggests that the fetus may be protected from allergic responses, at least in part, by controlling N addition.
Asunto(s)
Asma/genética , ADN Nucleotidilexotransferasa/genética , Animales , Asma/inmunología , Asma/patología , ADN Nucleotidilexotransferasa/inmunología , Modelos Animales de Enfermedad , Eliminación de Gen , Inmunidad Innata , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Ratones , Ratones Endogámicos BALB CRESUMEN
In the mouse thymus, invariant γδ T cells are generated at well-defined times during development and acquire effector functions before exiting the thymus. However, whether such thymic programming and age-dependent generation of invariant γδ T cells occur in humans is not known. Here we found that, unlike postnatal γδ thymocytes, human fetal γδ thymocytes were functionally programmed (e.g., IFNγ, granzymes) and expressed low levels of terminal deoxynucleotidyl transferase (TdT). This low level of TdT resulted in a low number of N nucleotide insertions in the complementarity-determining region-3 (CDR3) of their TCR repertoire, allowing the usage of short homology repeats within the germline-encoded VDJ segments to generate invariant/public cytomegalovirus-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). Furthermore, both the generation of invariant TCRs and the intrathymic acquisition of effector functions were due to an intrinsic property of fetal hematopoietic stem and precursor cells (HSPCs) caused by high expression of the RNA-binding protein Lin28b. In conclusion, our data indicate that the human fetal thymus generates, in an HSPC/Lin28b-dependent manner, invariant γδ T cells with programmed effector functions.
Asunto(s)
Feto/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Timocitos/inmunología , Timo/inmunología , Células Cultivadas , Niño , Preescolar , ADN Nucleotidilexotransferasa/inmunología , Femenino , Humanos , Lactante , Masculino , Proteínas de Unión al ARN/inmunologíaRESUMEN
AIMS: Very recent papers proposed a possible role for the expression of terminal deoxynucleotidyl transferase (TdT) in the tumourigenesis of gonadal and extragonadal germ cell-derived tumours (GCTs). Our multicentric study evaluated the magnitude of the immunoreactivity for TdT in GCTs, encompassing seminoma, dysgerminoma, mature teratoma and mixed GCTs. METHODS AND RESULTS: The histological series was stained with both monoclonal and polyclonal antibodies, yielding a positivity of 80% of cases with well-defined nuclear reactivity. A significant difference in staining intensity between monoclonal and polyclonal antibodies was observed (p=0.005). However, exploiting western blot and more innovative proteomic approaches, no clear-cut evidence of the TdT protein was observed in the neoplastic tissues of the series. CONCLUSIONS: Alternatively to the pathogenetic link between TdT expression and GCTs tumourigenesis, we hypothesised the occurrence of a spurious immunohistochemical nuclear cross-reaction, a well-known phenomenon with important implications and a possible source of diagnostic pitfalls in routine practice for pathologists.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Biomarcadores de Tumor/análisis , ADN Nucleotidilexotransferasa/análisis , Inmunohistoquímica , Neoplasias del Mediastino/enzimología , Neoplasias de Células Germinales y Embrionarias/enzimología , Neoplasias Ováricas/enzimología , Neoplasias Testiculares/enzimología , Biomarcadores de Tumor/inmunología , Reacciones Cruzadas , ADN Nucleotidilexotransferasa/inmunología , Femenino , Humanos , Italia , Masculino , Neoplasias del Mediastino/inmunología , Neoplasias del Mediastino/patología , Neoplasias de Células Germinales y Embrionarias/inmunología , Neoplasias de Células Germinales y Embrionarias/patología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Neoplasias Testiculares/inmunología , Neoplasias Testiculares/patologíaRESUMEN
The formation of nontemplated (N) regions during Ig gene rearrangement is a major contributor to Ab diversity. To gain insights into the mechanisms behind this, we studied the nucleotide composition of N regions within 29,962 unique human VHDJH rearrangements and 8728 unique human DJH rearrangements containing exactly one identifiable D gene segment and thus two N regions, N1 and N2. We found a distinct decreasing content of cytosine (C) and increasing content of guanine (G) across each N region, suggesting that N regions are typically generated by concatenation of two 3' overhangs synthesized by addition of nucleoside triphosphates with a preference for dCTP. This challenges the general assumption that the terminal deoxynucleotidyl transferase favors dGTP in vivo. Furthermore, we found that the G and C gradients depended strongly on whether the germline gene segments were trimmed or not. Our data show that C-enriched N addition preferentially happens at trimmed 3' ends of VH, D, and JH gene segments, indicating a dependency of the transferase mechanism upon the nuclease mechanism.
Asunto(s)
ADN Nucleotidilexotransferasa/inmunología , Reordenamiento Génico de Cadena Pesada de Linfocito B , Cadenas Pesadas de Inmunoglobulina , Región Variable de Inmunoglobulina , Adolescente , Adulto , Niño , Preescolar , Citosina/inmunología , ADN Nucleotidilexotransferasa/genética , Femenino , Guanosina/genética , Guanosina/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , MasculinoRESUMEN
B cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) of Salmonella enterica serovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/- and TdT-/- mice generated comparable antibody responses to Pneumovax23 and survived Streptococcus pneumoniae challenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/- or TdT-/- mice conferred protection. TdT+/- and TdT-/- mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity against S Typhi in vitro To test the protective immunity conferred by ViPS immunization in vivo, TdT+/- and TdT-/- mice were challenged with a chimeric Salmonella enterica serovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts for S Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/- and TdT-/- mice challenged with ViPS-expressing S Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.
Asunto(s)
ADN Nucleotidilexotransferasa/inmunología , Vacunas Neumococicas/inmunología , Polisacáridos Bacterianos/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Vacunas Tifoides-Paratifoides/inmunología , Factores de Edad , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Carga Bacteriana , ADN Nucleotidilexotransferasa/genética , Inmunización Pasiva , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Salmonella typhi/inmunología , Determinación de Anticuerpos Séricos Bactericidas , Streptococcus pneumoniae/inmunología , VacunaciónRESUMEN
The IKZF1 gene encodes the Ikaros protein, a zinc finger transcriptional factor that acts as a master regulator of hematopoiesis and a tumor suppressor in leukemia. Impaired activity of Ikaros is associated with the development of high-risk acute lymphoblastic leukemia (ALL) with a poor prognosis. The molecular mechanisms that regulate Ikaros' function as a tumor suppressor and regulator of cellular proliferation are not well understood. We demonstrated that Ikaros is a substrate for Casein Kinase II (CK2), an oncogenic kinase that is overexpressed in ALL. Phosphorylation of Ikaros by CK2 impairs Ikaros' DNA-binding ability, as well as Ikaros' ability to regulate gene expression and function as a tumor suppressor in leukemia. Targeting CK2 with specific inhibitors restores Ikaros' function as a transcriptional regulator and tumor suppressor resulting in a therapeutic, anti-leukemia effect in a preclinical model of ALL. Here, we review the genes and pathways that are regulated by Ikaros and the molecular mechanisms through which Ikaros and CK2 regulate cellular proliferation in leukemia.
Asunto(s)
Quinasa de la Caseína II/genética , Cromatina/inmunología , Factor de Transcripción Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linfocitos T/inmunología , Animales , Antineoplásicos/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/inmunología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Cromatina/química , Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , ADN Nucleotidilexotransferasa/genética , ADN Nucleotidilexotransferasa/inmunología , Regulación de la Expresión Génica , Humanos , Factor de Transcripción Ikaros/inmunología , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/inmunología , Naftiridinas/farmacología , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Fenazinas , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Transducción de Señal , Linfocitos T/efectos de los fármacos , Linfocitos T/patologíaRESUMEN
Multiparametric flow cytometry is an alternative approach to the polymerase chain reaction method for evaluating minimal residual disease in treatment protocols for primary acute lymphoblastic leukemia. Given considerable differences between primary and relapsed acute lymphoblastic leukemia treatment regimens, flow cytometric assessment of minimal residual disease in relapsed leukemia requires an independent comprehensive investigation. In the present study we addressed evaluation of minimal residual disease by flow cytometry in the clinical trial for childhood relapsed acute lymphoblastic leukemia using eight-color flow cytometry. The major challenge of the study was to reliably identify low amounts of residual leukemic cells against the complex background of regeneration, characteristic of follow-up samples during relapse treatment. In a prospective study of 263 follow-up bone marrow samples from 122 patients with B-cell precursor acute lymphoblastic leukemia, we tested various B-cell markers, adapted the antibody panel to the treatment protocol, and evaluated its performance by a blinded parallel comparison with the polymerase chain reaction data. The resulting eight-color single-tube panel showed a consistently high overall concordance (P<0.001) and, under optimal conditions, sensitivity similar to that of the reference polymerase chain reaction method. Overall, evaluation of minimal residual disease by flow cytometry can be successfully integrated into the clinical management of relapsed childhood acute lymphoblastic leukemia either as complementary to the polymerase chain reaction or as an independent risk stratification tool. ALL-REZ BFM 2002 clinical trial information: NCT00114348.
Asunto(s)
Biomarcadores de Tumor/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Antígenos CD/genética , Antígenos CD/inmunología , Antineoplásicos/uso terapéutico , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Biomarcadores de Tumor/inmunología , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Médula Ósea/patología , Preescolar , ADN Nucleotidilexotransferasa/genética , ADN Nucleotidilexotransferasa/inmunología , Citometría de Flujo/métodos , Expresión Génica , Humanos , Inmunofenotipificación , Lactante , Neoplasia Residual , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , RecurrenciaRESUMEN
BACKGROUND: There is paucity of detailed studies of adult T cell acute lymphoblastic leukemia (T-ALL) in developing countries reflecting the condition of these patients including clinical and biological features. OBJECTIVE: This study was carried out to analyze the immunophenotypic characteristics of 40 Moroccan patients with T-ALL and its association with biological and clinical features. PATIENTS AND METHODS: Between 2006 and 2009, 130 adult patients diagnosed with acute lymphoblastic leukemia (ALL) were immunophenotyped by 3-color flow cytometry using a panel of monoclonal antibodies. Cases presenting features of a T-lineage phenotype were subjected to detailed analysis including immunophenotypic, clinical and biological parameters. RESULTS: Proportion of T-ALL among ALL Moroccan patients was 31.0%. Median age of patients was 28 years. Twenty-nine patients were females and 11 were males. 45.0% of patients (18/40) had features of immature T-ALL stages (pro-T and pre-T ALL), 30.0% (12/40) of CD1a+ cortical T-ALL stage and 25.0% (10/40) had a characteristic phenotype of medullary T-ALL. The frequencies of progenitor cell markers CD10, CD34 and TdT expression were 14.0; 57.5% and 50.0% respectively. The aberrant expression of B lineage associated antigen CD79a were positive in 20.5% of the cases and the aberrant expression of myeloid antigens CD13 and/or CD33 was found in 22 (55.0%) cases. No significant association was encountered between TdT, CD34 or myeloid antigens positivity and high risk features at presentation as age, sex, and white blood cells. However, myeloid antigens (CD13 and/or CD33) was significantly associated with T-cell maturation stages (p = 0.009). CONCLUSION: To the best of our knowledge, this is the first report from North Africa of immunophenotypic study on adult T-ALL. Our findings indicate that the proportion of T-ALL among ALL in Morocco is similar to that reported in others Mediterranean countries like France and Italy and that myeloid-associated antigens expression is frequently associated with immature immunophenotype.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Adolescente , Adulto , Anciano , Antígenos CD/análisis , Antígenos CD/inmunología , ADN Nucleotidilexotransferasa/análisis , ADN Nucleotidilexotransferasa/inmunología , Femenino , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Marruecos/epidemiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiología , Adulto JovenRESUMEN
BACKGROUND: The loss of CD45, the leukocyte-common antigen, has been described in rare cases of large B-cell lymphoma (LBCL) subtypes with extranodal involvement by immunohistochemical methods. Here we report a case of a patient with LBCL, with no extranodal lesions, which is CD45 negative by flow cytometry (FC) immunophenotyping. METHODS: Immunophenotyping and DNA content analysis was performed by multiparametric FC on lymph node and bone marrow aspirate obtained from a 65 year old male patient. RESULTS: Malignant B-lymphocytes were CD5-, CD10+/++, CD11c-, CD19+, CD20+/++, CD23-, CD34-, CD38-/+, CD45-, CD79b++/+++, BCL2 overexpressed, FMC7++, IgM++/+++, TdT- with Lambda light chain restriction. This pathological cellular population showed near-diploid DNA content, with a high proliferate rate. CONCLUSIONS: To our knowledge, we describe the first case of a CD19+ B-cell non-Hodgkin lymphoma without expression of CD45 detected by FC, and the first case without extranodal involvement presentation. This case is reported not only because it is a rare one but also to raise awareness of FC users of its correct diagnosis.
Asunto(s)
Citometría de Flujo/métodos , Antígenos Comunes de Leucocito/metabolismo , Linfoma de Células B Grandes Difuso/diagnóstico , Anciano , Antígenos CD/análisis , Antígenos CD/inmunología , Linfocitos B/inmunología , Biopsia , Médula Ósea/inmunología , Proliferación Celular , ADN Nucleotidilexotransferasa/análisis , ADN Nucleotidilexotransferasa/inmunología , Glicoproteínas/análisis , Humanos , Inmunoglobulina M/análisis , Inmunofenotipificación/métodos , Antígenos Comunes de Leucocito/análisis , Antígenos Comunes de Leucocito/inmunología , Ganglios Linfáticos/inmunología , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/patología , MasculinoRESUMEN
The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT(-/-)) and wild-type (TdT(+/+)) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT(-/-) cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP(19)CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT(-/-) bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgM(a) and congenic TdT-sufficient CB17 IgM(b) bone marrow were placed in competition. TdT(-/-) cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens.
Asunto(s)
Activación de Linfocitos/inmunología , Animales , Antígenos CD19/inmunología , Linfocitos B/inmunología , Médula Ósea/inmunología , Trasplante de Médula Ósea/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , ADN Nucleotidilexotransferasa/genética , ADN Nucleotidilexotransferasa/inmunología , Dextranos/inmunología , Enterobacter cloacae/inmunología , Ficoll/inmunología , Haptenos/inmunología , Inmunidad Humoral , Inmunoglobulina M/inmunología , Ratones , Ratones Endogámicos BALB C , Muramidasa/inmunología , Fosforilcolina/inmunología , Picratos/inmunología , Bazo/inmunología , Streptococcus pneumoniae/inmunologíaRESUMEN
Immunophenotyping is one of the independent prognostic factors in acute myeloid leukemia (AML). Relevance of immunophenotypes to prognostic subgroups of age, white blood cells (WBC), platelet count, and cytogenetics in de novo AML was comprehensively investigated in this study for the first time. Human leukocyte antigen (HLA)-DR and CD14 expression associated with the elderly, highest WBC count, and unfavorable-risk cytogenetics; CD4, CD7, and CD11b expression correlated with highest WBC count and unfavorable-risk cytogenetics; CD64 expression was associated with higher WBC count while that of CD13 was associated with lower platelet count; CD22, CD34, CD123, and terminal deoxynucleotidyl transferase (TdT) expression correlated with unfavorable-risk cytogenetics; CD5 expression was associated with normal platelet count while that of CD19 was associated with children and favorable-risk cytogenetics; CD117 expression was associated with low WBC and lower platelet counts; myeloperoxidase (MPO) expression correlated with lower platelet count; and MPO and glycophorin A (Gly-A) expression was associated with lower WBC count and favorable-risk cytogenetics. The results of the relevance analysis revealed the distribution characteristics of antigen expression in different AML prognostic subgroups. The majority of antigens associated with good or poor prognostic subgroups were in accordance with the previous reports of correlation of expression of these antigens with prognosis. Antigens associated with good (or poor) prognostic subgroups were defined as good (or poor)-risk antigens.
Asunto(s)
Antígenos CD/inmunología , ADN Nucleotidilexotransferasa/inmunología , Inmunoglobulina G/sangre , Leucemia Mieloide Aguda/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/sangre , Distribución de Chi-Cuadrado , Niño , Preescolar , ADN Nucleotidilexotransferasa/sangre , Femenino , Humanos , Inmunofenotipificación , Lactante , Leucemia Mieloide Aguda/sangre , Masculino , Persona de Mediana Edad , Pronóstico , Adulto JovenRESUMEN
An understanding of Ab responses to polysaccharides associated with pathogenic microorganisms is of importance for improving vaccine design, especially in neonates that respond poorly to these types of Ags. In this study, we have investigated the role of the lymphoid-specific enzyme TdT in generating B cell clones responsive to alpha-1,3 dextran (DEX). TdT is a DNA polymerase that plays a major role in generating diversity of lymphocyte AgRs during V(D)J recombination. In this study, we show that the DEX-specific Ab response is lower, and the dominant DEX-specific J558 idiotype (Id) is not detected in TdT(-/-) mice when compared with wild-type (WT) BALB/c mice. Nucleotide sequencing of H chain CDR3s of DEX-specific plasmablasts, sorted postimmunization, showed that TdT(-/-) mice generate a lower frequency of the predominant adult molecularly determined clone J558. Complementation of TdT expression in TdT(-/-) mice by early forced expression of the short splice variant of TdT-restored WT proportions of J558 Id+ clones and also abrogated the development of the minor M104E Id+ clones. J558 Id V(D)J rearrangements are detected as early as 7 d after birth in IgM-negative B cell precursors in the liver and spleen of WT and TdT-transgenic mice but not in TdT(-/-) mice. These data show that TdT is essential for the generation of the predominant higher-affinity DEX-responsive J558 clone.
Asunto(s)
Linfocitos B/inmunología , ADN Nucleotidilexotransferasa/inmunología , Dextranos/inmunología , Animales , Células Clonales/inmunología , ADN Nucleotidilexotransferasa/deficiencia , ADN Nucleotidilexotransferasa/genética , Reordenamiento Génico , Genes de Inmunoglobulinas , Inmunidad Humoral , Región de Unión de la Inmunoglobulina/genética , Ratones , Ratones Noqueados , Polisacáridos/inmunologíaAsunto(s)
Anticuerpos Monoclonales/biosíntesis , ADN Nucleotidilexotransferasa/biosíntesis , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Anticuerpos Monoclonales/inmunología , Linfoma de Burkitt/patología , ADN Nucleotidilexotransferasa/inmunología , Diagnóstico Diferencial , Femenino , Citometría de Flujo , Humanos , Cadenas Ligeras de Inmunoglobulina/inmunología , Hibridación Fluorescente in Situ , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunologíaRESUMEN
We have identified a distinctive lymphoid-restricted progenitor population in adult mouse bone marrow based on a unique c-Kit(-)Sca-1(high)Flt3(+) AA4(+) surface phenotype. These cells are highly lymphoid biased and rapidly generate B and T cells after adoptive transfer. However, whereas previously described lymphoid progenitors such as common lymphoid progenitors express TdT and relatively high levels of RAG2, and are enriched for cells with an active V(D)J recombinase, Flt3(+) AA4(+) cells within the c-Kit(-)Sca-1(high) bone marrow fraction are TdT(-), are RAG2(low), and do not display evidence for ongoing or past recombinase activity. Furthermore, unlike common lymphoid progenitors that readily generate B cells upon stimulation with IL-7, c-Kit(-)Sca-1(high)Flt3(+) precursors do not express abundant levels of the IL-7R, and require costimulation with Flt3 ligand and IL-7 to generate B cells in vitro. Moreover, these findings suggest that hematopoietic stem cells in adults generate an array of lymphoid-biased progenitor populations characterized by distinct gene expression and cytokine response profiles.
Asunto(s)
Antígenos Ly , Linfocitos B/citología , Diferenciación Celular/fisiología , Células Progenitoras Linfoides/citología , Proteínas de la Membrana , Proteínas Proto-Oncogénicas c-kit , Animales , Linfocitos B/inmunología , Células Cultivadas , ADN Nucleotidilexotransferasa/inmunología , Proteínas de Unión al ADN/inmunología , Interleucina-7/inmunología , Células Progenitoras Linfoides/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Noqueados , Receptores de Complemento/inmunología , Receptores de Interleucina-7/inmunología , VDJ Recombinasas/inmunología , Tirosina Quinasa 3 Similar a fms/inmunologíaRESUMEN
The action of TdT on mouse TCR genes accounts for approximately 90% of T cell repertoire diversity. We report that in TdT-/- mice, total T(CD8+) responses to influenza and vaccinia viruses are reduced by approximately 30% relative to wild-type mice. We find that T(CD8+) responses to three subdominant influenza virus determinants are reduced to background values in TdT-/- mice while responses to three immunodominant determinants undergo a major reshuffling. A similar reshuffling occurs in T(CD8+) responses to immunodominant vaccinia virus determinants, and is clearly based on broad differences in TCR family usage and CDR3 length between wild-type and TdT-/- mice. These findings demonstrate that TdT plays a critical role in the magnitude and breadth of anti-viral T(CD8+) responses toward individual determinants and suggests that germline TCR repertoire bias toward the most dominant determinants is a major factor in establishing immunodominance hierarchies.
Asunto(s)
Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , ADN Nucleotidilexotransferasa/inmunología , ADN Nucleotidilexotransferasa/metabolismo , Animales , Presentación de Antígeno/inmunología , Antígenos Virales/inmunología , ADN Nucleotidilexotransferasa/deficiencia , ADN Nucleotidilexotransferasa/genética , Femenino , Virus de la Influenza A/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunología , Virus Vaccinia/inmunologíaRESUMEN
We have used selected rabbit anti-human polyclonal antibodies as an example of useful and easily available tools for studies on immune system structure and development in important veterinary species, many of which also represent animal models in biomedicine. The cocktail of anti-human Igkappa-FITC/anti-Iglambda-RPE F(ab')(2) fragments was used for two-colour and, in combination with the cross-reactive anti-CD79alpha monoclonal antibody HM-57, for three-colour flow cytometry of canine, feline, bovine and porcine peripheral B-cells. A possible application of such immunoreagents in studies on primary B-cell differentiation has been suggested in pigs; the same approach can be used in other species of interest. Rabbit anti-human lactoferrin-FITC F(ab')(2) fragment was used for visualizing neutrophils in dogs, pigs and cattle and an application for two-colour immunophenotyping of canine granulocyte subsets has been designed. Affinity isolated rabbit anti-human CD3 and anti-human TdT have been shown to represent a ready-to-use tool for in situ studies on primary T-lymphopoiesis in pigs with possible extensions both to the B-lineage development in pigs and other animal models. Altogether, our study show that carefully selected polyclonal antibodies available on the market may possess broad cross-reactivity with important applications in veterinary research.
Asunto(s)
Sueros Inmunes/inmunología , Sistema Inmunológico/fisiología , Animales , Antígenos CD/análisis , Antígenos CD/inmunología , Gatos , Bovinos , Reacciones Cruzadas , ADN Nucleotidilexotransferasa/análisis , ADN Nucleotidilexotransferasa/inmunología , Perros , Citometría de Flujo , Humanos , Cadenas Ligeras de Inmunoglobulina/análisis , Cadenas Ligeras de Inmunoglobulina/inmunología , Inmunohistoquímica , Conejos , PorcinosRESUMEN
AIMS: The diagnosis of primitive hematologic malignancies depends on a panel of monoclonal antibodies which is growing over time. The distinction between immature (lymphoblastic lymphoma/acute lymphoblastic leukaemia) and mature lymphoma is sometimes difficult. In this study, we evaluated anti-TdT antibody in the diagnosis and classification of these proliferations. MATERIALS AND METHODS: 13 lesions were examined by immunohistochemistry: 4 B and T lymphoblastic lymphomas, 2 Burkitt's lymphomas, 5 B and T acute lymphoblastic leukemias and 2 acute monoblastic leukemias. RESULTS: TdT expression is specific of immature lymphoid proliferations (T or B lymphoblasts). TdT is not expressed by mature B or T cell lymphomas such as Burkitt's lymphomas. Significant numbers of cases of acute myeloblastic leukemias are TdT positive but could be easily distinguished from lymphoblastic proliferations. CONCLUSION: Anti-TdT antibody represents a useful marker for differentiating lymphoma/acute lymphoblastic leukemia from other lymphomas. This marker, available in routine diagnosis should be systematically included in the panel of antibodies used for immunophenotyping hematologic malignancies.
Asunto(s)
Anticuerpos , ADN Nucleotidilexotransferasa/análisis , ADN Nucleotidilexotransferasa/inmunología , Neoplasias Hematológicas/diagnóstico , Adolescente , Adulto , Anciano , Linfoma de Burkitt/diagnóstico , Niño , Diagnóstico Diferencial , Femenino , Neoplasias Hematológicas/clasificación , Neoplasias Hematológicas/enzimología , Humanos , Inmunohistoquímica , Inmunofenotipificación , Leucemia Monocítica Aguda/diagnóstico , Leucemia-Linfoma de Células T del Adulto/diagnóstico , Linfoma de Células B/diagnóstico , Linfoma de Células T/diagnóstico , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnósticoRESUMEN
It has been suggested that recombination activating gene (RAG)-dependent revision of the immunoglobulin genes in germinal centres may contribute to local production of autoantibodies in Hashimoto thyroiditis (HT) and rheumatoid arthritis (RA). To test this hypothesis we examined HT and RA tissues for expression of RAG and terminal deoxynucleotidyl transferase (TdT) in situ. Paraffin-embedded tissues from 19 HT patients and from 20 RA patients were subjected to immunohistochemistry using TdT-specific antibodies. Expression of the RAGs was studied by in situ hybridisation. Tonsil sections were used as a control. Expression of TdT and RAGs was detected in extrafollicular lymphocytes in control tonsil sections. By contrast, only rare TdT-expressing cells were identified in 11 of 19 HT and in 2 of 20 RA samples. Germinal centre B-cells were consistently TdT- and RAG-negative. These results suggest that local RAG-dependent receptor revision in germinal centres is unlikely to contribute to production of autoantibodies in HT and RA. The presence of TdT-positive extrafollicular cells may represent an influx of immature cells in the context of chronic immune stimulation.
Asunto(s)
Artritis Reumatoide/inmunología , ADN Nucleotidilexotransferasa/genética , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Linfocitos/patología , Tiroiditis Autoinmune/inmunología , Adolescente , Adulto , Anciano , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Autoanticuerpos/inmunología , Diferenciación Celular/inmunología , ADN Nucleotidilexotransferasa/inmunología , Proteínas de Unión al ADN/inmunología , Femenino , Centro Germinal/inmunología , Centro Germinal/patología , Proteínas de Homeodominio/inmunología , Humanos , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Nucleares , Tiroiditis Autoinmune/genética , Tiroiditis Autoinmune/patologíaRESUMEN
CONTEXT: Acute leukemia displays characteristic patterns of surface antigen expression (CD antigens), which facilitate their identification and proper classification and hence play an important role in instituting proper treatment plans. In addition to enzyme cytochemical analysis, multiparameter flow cytometric analysis has become commonplace in most laboratories for that purpose. The essential role and caveats of flow cytometry in that regard, however, have received little scrutiny. OBJECTIVE: To evaluate the expression of commonly used immunomarkers and patterns in various acute leukemias to help define the best use and role of multiparameter flow cytometry in the diagnosis and proper classification of acute leukemias. DESIGN: We have retrospectively analyzed the immunophenotypic data from 508 de novo adult and pediatric acute leukemia patients, as studied using multiparameter flow cytometry in addition to routine morphologic and enzyme cytochemical analysis. Cytogenetic and/or molecular data were correlated in all 41 cases of acute promyelocytic leukemia (APL) and in 203 other cases of acute leukemia where those data were available. We have also determined the positive and negative predictive values of a combined CD34 and HLA-DR expression pattern for the differentiation of APL from other myeloid leukemias. RESULTS: In acute myeloid leukemia (AML) other than APL, expression of CD34 was seen in 62% and expression of HLA-DR in 86% of all cases. Twenty-six (10%) of 259 cases of non-APL AML were negative for both CD34 and HLA-DR as opposed to 33 (80%) of 41 cases of APL. None of the cases of APL were positive for both CD34 and HLA-DR in contrast to 149 (58%) of 259 cases of non-APL AML. Fifty-three cases were found to be examples of minimally differentiated AML (AML M0) based on the lack of expression of cytoplasmic CD3 and cytoplasmic CD79a and expression of one or more myelomonocytic-associated antigens and/or myeloperoxidase. Expression of CD20 was seen in 40 (24%) of 168 cases of precursor B-cell acute lymphoblastic leukemia (pB-ALL) and 52 (29%) lacked CD34 expression. Five of 180 cases of pB-ALL and 2 cases of precursor T-cell ALL (pT-ALL) were negative for terminal deoxynucleotidyl transferase (TdT). Aside from cytoplasmic CD3, CD5 and CD7 were the most sensitive antigens present in all 21 cases of pT-ALL. CD33 was more sensitive but less specific than CD13 for myeloid lineage. CONCLUSION: Aside from identification of blasts, flow cytometry was found to be especially useful in the correct identification of AML M0, differentiation of APL from AML M1/M2, and correct identification of TdT-negative ALL and unusual variants, such as transitional B-cell ALL and undifferentiated and biphenotypic acute leukemias.
Asunto(s)
Citometría de Flujo , Inmunofenotipificación , Leucemia/clasificación , Enfermedad Aguda , Adulto , Antígenos CD34/inmunología , Antígenos CD34/metabolismo , Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Análisis Citogenético , ADN Nucleotidilexotransferasa/inmunología , ADN Nucleotidilexotransferasa/metabolismo , Antígenos HLA-DR/inmunología , Antígenos HLA-DR/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Leucemia/diagnóstico , Leucemia/metabolismo , Megacariocitos/metabolismo , Peroxidasa/inmunología , Peroxidasa/metabolismo , Proteínas Proto-Oncogénicas c-kit/inmunología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Estudios RetrospectivosRESUMEN
The short splice variant of mouse terminal deoxynucleotidyl transferase (TdTS) catalyzes the addition of nontemplated nucleotides (N addition) at the coding joins of B cell and T cell antigen receptor genes. However, the activity and function of the long isoform of TdT (TdTL) have not been determined. We show here, in vitro and in vivo, that TdTL is a 3'-->5' exonuclease that catalyzes the deletion of nucleotides at coding joins. These findings suggest that the two TdT isoforms may act in concert to preserve the integrity of the variable region of antigen receptors while generating diversity.